6/26/01

Lecture #2 - Bacterial Transcription

Central dogma of molecular biology: DNA--> RNA--> protein

transcription: DNA--> RNA

The level of initiation of transcription is the most important control point for gene regulation in both prokaryotes and eukaryotes

Unicellular organisms (e.g. bacteria, yeast)have to respond to environmental factors (temperature, pH, salt concentration, carbon source, nitrogen source, toxins) and they do this by turning turning on and off genes in response to these factors.

Multicellular eukaryotes turn genes on and off in different circumstances.

Spatial control (e.g. only in liver or only in brain)
Temporal control - genes on only at certain times (e.g. embryonic development or adulthood)

Bacteria
Eukaryotes

RNA polymerase

Single RNA polymerase synthesizes mRNA, rRNA, and tRNA

RNA pol I - rRNA

RNA pol II - mRNA

RNA pol III - small RNA's

RNA processing

Little or no processing; primary transcript is the final RNA

Widespread introns; introns removed post-transcriptionally; lot more RNA processing than in bacteria

Genes per transcript

Many genes present on same mRNA (polycistronic)

Operon: cluster of genes

One gene per mRNA (monocistronic)

RNA is transcribed off the template (antisense) strand of DNA; it is identical in sequence to the coding (sense) strand of DNA

This RNA is then translated into a polypeptide; however, not all of it is translated. The ORF (Open Reading Frame) is the region that actually encodes the protein. The non-encoding parts of the RNA are called UTR's (Untranslated Regions). At the 5' end of the RNA is the 5' UTR and at the 3' end is the 3' UTR.

The double stranded DNA has a promoter, which is usually at the 5' end of the gene and is found upstream of the transcription start site; the DNA also has a terminator, where transcription is terminated.

This DNA fragment containing the promoter, the transcription start site, the gene, and the terminator is called the transcription unit.

Promoters usually have negative numbers to indicate that they are "upstream" of the start site.

RNA polymerase (RNAP)

To synthesize RNA, ribonucleotide triphosphates are needed.
The a phosphate is incorporated into the RNA.
RNA polymerase catalyzes the following reaction: RNA + NTP --> RNA + 1 + P-P (1 denotes the extension of the RNA by 1 nucleotide; the P-P is a pyrophosphate)

Two characteristics of RNA polymerase:

RNA synthesis is always in the 5' to 3' direction
RNA polymerase can synthesize RNA "de novo"; in other words, it doesn't require a primer to synthesize RNA; it only needs a template. This differs from DNA polymerase, which requires a primer for DNA synthesis in all cases.

In E. Coli, there are 5 subunits in RNA polymerase, denoted by a ₂b b's

The a ₂b b' subunits comprise the "core RNA polymerase," which is capable of carrying out elongation, but not initiation.
The a ₂b b's subunits make up the holoenzyme, which can carry out elongation AND initiation (i.e. it can recognize the promoter)
The s subunit recognizes the promoter
The molecular weight of the entire E. Coli holoenzyme is 465,000 Daltons.

Why is the RNA polymerase so large?

The T7 bacteriophage has a smaller RNA polymerase.

T7 encodes its own RNA polymerase, which is a single subunit protein 90 kDaltons
T7 RNA polymerase is better and faster than the E. coli RNA polymerase
It is more processive (i.e. it falls off the DNA template less often than E. coli's RNA polymerase)

It is uncertain how the large RNA polymerase helps; it may be that with more subunits, can control gene regulation and transcription can be tweaked

The four stages of transcription are:

template recognition
initiation
elongation
termination.

Recognition

RNA polymerase recognizes certain sequences in the promoter.
RNA polymerase binds to duplex DNA, forming the "closed complex" , i.e. DNA is still base paired in promoter.
Isomerization - some of the base pairs are melted, forming the "open complex" - a region in which the DNA is not base paired
The formation of the open complex is a discrete step from the formation of the closed complex.

Initiation

Chains of 2-9 nucleotide long RNA are synthesized and released - abortive initiation, but RNA polymerase still sits at the promoter.
RNA polymerase then clears the promoter and begins making longer RNAs
When the RNA polymerase leaves the promoter, the s subunit, which is not required for elongation, dissociates from the complex. Core RNAP synthesizes RNA.

Elongation

The core RNA polymerase then moves down the template and unwinds the DNA, forming a transcription bubble, a region of DNA that is not base paired and where RNA is synthesized.

Termination

At the termination sequence, the RNA polymerase dissociates from the template and the RNA is released.

All these steps take place in eukaryotes; the only thing that differs is the machinery used (i.e. it is more complex).

Rate of RNA polymerase: 40 nucleotides synthesized per second

Rate of translation in bacteria: 15 amino acids synthesized per second (15 amino acids = 45 nucleotides)

The transcription and translation rates are about the same because they occur simultaneously in bacteria. (i.e. they are "coupled"). As soon as an RNA is synthesized, ribosomes can bind to the RNA and translate it.

In eukaryotes, transcription and translation are not coupled. Transciption takes place in the nucleus, while translation takes place in the cytoplasm.

Rate of DNA polymerase: 800 nucleotides per second.

Supercoiling of DNA during transcription

The DNA double helix is a coil with about 10 base pairs per turn.
Can either overwind (extra turns; (+) supercoiled) or underwind (fewer turns; (-) supercoiled) DNA
Transcription may generate overwound DNA in front of the RNA polymerase and underwound DNA behind the RNA polymerase.

Enzymes that affect supercoiling:

Helicases unwind double stranded DNA without breaking the strands.
Topoisomerases unwind or overwind DNA by cutting open and rejoining strands of DNA

Promoters

Promoters are sequences recognized by RNA polymerase and tell RNA polymerase where to initiate transcription.

"not all promoters are created equal."

Some promoters can be strong - high levels of transcription; high affinity for RNA polymerase
Some promoters can be weak - low levels of transcription; low affinity for RNA polymerase
The sequence of the promoter distinguishes strong promoters from weak promoters

Strong promoters in E. coli

5' -------------------TTGACA-------------17 bp-----------TATAAT-----7 bp-----purine 3 '

-35: TTGACA

-10: TATAAT

The RNA start site is a purine 90% of the time
Strong promoters tend to have matches to these consensus sequences
Between -55 and -35, there is an AT rich region called the "UP" element
The "UP" element is present in the strongest promoters, such as those for rRNAs

Promoter mutations

Can convert strong promoter to weak promoter or vice versa by changing the sequence

"up" mutation: start with a weak promoter--> stronger promoter, i.e. poor match to consensus sequence--> closer match to consensus sequence
"down" mutation: good match--> poorer match, i.e. strong match to consensus sequence--> weaker match to consensus sequence

-10 and - 35 consensus sequences

-35 mutations affect the rate of closed complex formation
The s subunit recognizes the - 35 region
A mutation in the -10 region affects the rate of the open complex formation
The -10 region is AT rich, which makes it easier to melt apart than a GC rich region (There are only two hydrogen bonds between A and T; there are three hydrogen bonds between G and C.)
Deletions and insertionscan change the spacing in the 17 bp and 7 bp regions

How to control transcription

Promoter strength - can change degree of similarity to consensus sequence
Presence/absence of regulatory proteins - activators and repressors
Alternative sigma factors
- s ⁷⁰ in the holoenzyme recognizes the -35 and -10 sequences
- Other s factors recognize different sequences
- s ⁵⁴ recognizes consensus sequences at - 24 and -12
- s ⁵⁴ is not sufficient to direct transcription; positive activation also required

Biochemistry and RNAP / promoter interaction

To study the interaction of RNA polymerase with DNA, want to know where RNA polymerase binds in the promoter?

How determine?? Footprint experiment

Need:

a purified form of the protein to be studied
DNA for the protein to bind to

The footprint experiment also requires labeled DNA, usually radioactively labeled at one end of one strand

How to label DNA

Treat DNA with alkaline phosphatase which removes the phosphates and converts the 5' phosphates into -OH groups.
Treat with T4 polynucleotide kinase and with g -ATP labeled with ³²P.
This gives DNA labeled at one end of BOTH strands.
Cut with restriction enzyme to yield DNA labeled at one end of one strand.

Footprint experiments are also called DNAseI protection experiments

Steps in DNAseI footpring experimentBegin with labeled DNA

Bind protein to DNA
Treat complex with DNAaseI which randomly cuts DNA. Use limiting amount of DNAaseI so that you cut on average each DNA molecule only once
DNAaseI will not cut where protein is bound.
Denature DNA and knock off protein.
Will only see fragments that have the labeled end - unlabeled fragments won't be visible on the gel.
Run DNA on a denaturing polyacrylamide gel which has a resolution of 1 base pair.
Region on gel with no bands = region where protein binds.
Do control with DNA and DNAaseI, but no protein, to compare control with experimental results; in the control, you may not see a perfect, regular "ladder"; DNAaseI cuts at almost every position but it prefers certain positions.

Chemical Footprint

DNAseI is an enzyme of about 50 kDaltons; because it is so large, it cannot distinguish regions loosely bound to RNA polymerase from regions strongly bound to RNA polymerase.

Chemical footprints use small chemicals such as DMS (dimethyl sulfate), which is only several hundred daltons; these small chemicals can diffuse into places where DNAseI cannot. Thus chemicals can distinguish close contact points from regions where DNA-RNA polymerase association is weak

Methylation interference

Methylate DNA at specific positions.
Bind protein to DNA.
Look for places where methylation PREVENTS binding.
This tells you the positions that MUST be contacted for protein to bind.

Many experiments have been done to look at interaction of RNA polymerase with promoter - Fig. 9.17 summarizes.

DNAaseI footprint - 50 to +20
Genetic approach and biochemical approach leads to the same answer: that - 35 and - 10 are important for interaction of RNAP and promoter DNA and for promoter function.
Can do two footprint experiments on separate strands
More contact points on coding strand, indicating that proteins can interact differentially with two strands of DNA.

Transcription Termination

In E. coli, there are two types of termination:

rho independent / intrinsic (more common)
rho dependent (rarer)

rho independent

Termination is dependent on sequences in the RNA that has been transcribed; depends on both the primary and secondary structure of RNA.
In the RNA, there can be an inverted repeat (e.g. GGG---- CCC)
A stem loop (secondary structure) can form, pausing the RNA polymerase.
There is usually a GC rich region in the stem loop because the G-C bond is more stable than an A-U bond (3 H-bonds rather than 2).
The stem loop is followed by a U-rich region, which is complementary to an A-rich region in the DNA template strand.
However, the deoxyribo-A base paired to ribo-U interaction is unstable.
When 1) a stem loop forms, pausing RNA polymerase, and 2) the U-rich region (which is unstably bound to the template A-rich region) is present, termination occurs, i.e. the RNA dissociates and RNAP falls off the template.

rho dependent

Rarer than rho independent
This type of termination was discovered two different ways: it was discovered genetically and biochemically.
It was studied biochemically by people doing in vitro transcription
They mixed RNA polymerase with NTPs, template DNA (containing a promoters, operon, 5'-3' UTRs etc.) and in the test tube, RNA was produced.
However, they realized that certain genes were unable to terminate transcription in vitro.
They could get these genes to terminate if they added an E. coli protein extract
This suggested that some E. coli protein could bring about termination of transcription
They used the in vitro transcription assay to purify the protein with the terminating activity and found it to be a single protein, which they called the rho protein.

rho protein

The rho protein is 46 kDaltons and has various activities
It has RNA dependent ATPase activity, meaning it can hydrolyze ATP when bound to RNA. ATP hydrolysis may allow rho to move on RNA.
It has helicase activity and can unwind duplex nucleic acids.
It is an RNA binding protein rich in the basic amino acids (Lys, Arg, His). (Positively charged amino acids are found in DNA binding proteins because these R groups facilitate binding to DNA, which is negatively charged (from its phosphate groups on the backbone.)
rho is active as a hexamer (6 subunits).
rho recognizes a C-rich G-poor region in RNA.
It is postulated that rho's ATPase activity allows it to move in a 5' to 3' direction on RNA.
rho catches up to the transcription machinery (i.e. RNAP), and rho then disrupts the interaction between RNA and DNA with its helicase activity, which terminates transcription (i.e. the RNA dissociates and RNAP falls off the template).